Tandem mass spectrometry is a powerful analytical technique which is used for structural analysis of chemical species, as well as for the specific detection of known targeted compounds in the presence of many other compounds, or in samples which contain a wide variety of endogenous species which otherwise would obscure the presence of the compound of interest.
Mass spectrometry is a known instrumental technique in which compounds to be analyzed are first converted to ions (or, if already in the form of ions, are separated from the surrounding liquid), and then separated or filtered according to their mass-to-charge ratio (m/z), before being detected and counted with an ion or current detector. The output of such analysis is usually a mass spectrum in which the signal at each mass-to-charge value is proportional to the concentration of each species which has that m/z. Many modern ionization techniques (for example, electrospray and atmospheric chemical pressure ionization) form ions which are indicative only of the molecular weight of the species. Since there can be many different compounds of different structure but the same molecular weight, the mass value is only of moderate specificity in the analysis of an unknown species. In addition, if more than one species of the same m/z value is present in a mixture, then the signal will be the sum of the responses of both species together, and the individual concentration of each species cannot be unambiguously determined without use of another separation technique that does distinguish between the two species, such as chromatography (which separates species based on their elution time from a column) or other chemical separation method.
Tandem mass spectrometry is a technique in which ions of selected m/z can be fragmented at a controlled energy, usually by collisions with a low density gas. By selecting a narrow m/z range (eg. 1 amu wide) to be transmitted into the collision cell, and recording the mass spectrum of fragment ions by means of a second mass spectrometer placed after the collision cell, a tandem mass spectrum or mass fingerprint of the precursor ion is produced. This technique of fragmentation of a selected ion mass is called MS/MS. The process of fragmentation in a low density gas is called collisionally activated dissociation (CAD).
The MS/MS spectrum shows fragments of the precursor ion which are characteristic of its structure. The MS/MS spectrum of an unknown compound can reveal information about its structure, and hence something about the identity of the compound. Even if the structure of the compound cannot be deduced from the MS/MS spectrum, the spectrum is at least a fingerprint which identifies the compound with much less ambiguity than does just the molecular weight. This fingerprint can be used to search for the presence of the compound in a complex mixture, or to confirm the presence of a specific compound whose MS/MS spectrum has been previously determined. “Libraries” of MS/MS spectra can be constructed and used to compare against unknown spectra in order to perform automated identification.
Structurally similar compounds often fragment in a similar fashion. Thus if one compound is related to another by having a methyl group substituted for a hydrogen atom, it is likely that the MS/MS spectra of the two compounds would have many fragments in common, even though the molecular weights differ by 14 Daltons. This relationship can provide a powerful tool to search for the presence of related compounds in complex mixtures, by searching for fragmentation patterns which have many peaks in common, or which have at least one peak in common. In other cases, the m/z of certain fragment ions will differ from that of the precursor ion by a fixed value, for example 18 units, indicating that both precursors lose the same neutral species during CAD. This provides another way of searching for the presence of related compounds in a complex mixture.
Another widely used advantage provided by tandem mass spectrometry is that if the instrument is tuned to pass or detect only specific product ions of specific precursor ion masses, then this can be used to screen complex samples for the presence of known compounds which have the selected precursor ion m/z and which form the selected product ion or ions. For example, it is known that the drug Reserpine (MW 608) forms a precursor ion of m/z 609 in an electrospray ion source, and that under CAD, some products of m/z 195 and 174 are formed. Therefore, in order to detect the presence of Reserpine in a sample (such as urine or blood serum), a tandem mass spectrometer can be tuned to pass only ions of m/z 609 into the collision cell, and to pass only ions of m/z 195 or 174 to the ion detector. Thus if a signal is received at both 195 and 174, there is little doubt that the target compound is present. The compound is identified by both the precursor ion mass (609) and the product ion masses (195 and 174). If only a single mass spectrometer were used to detect the presence of any ion of m/z 609, then the analysis would be more ambiguous, since many different compounds form ions of m/z 609. However, very few of these, (besides Reserpine) would form products of m/z 174 and 195.
Tandem mass spectrometers are therefore widely used to analyze complex samples for the presence of specific target compounds, and to measure how much of the target compound is present by recording the intensity of the ion signal at the corresponding precursor/product masses. For example, tandem mass spectrometers are commonly used for the analysis of biological fluids (such as blood and urine) for the presence of drugs and their metabolites. In cases where the targeted compounds are known, and the requirement is only to detect the presence and quantity of the drug, then the instrument is tuned to only transmit and respond to the specific precursor/product ion (this is called the multiple-reaction-monitoring or MRM mode). In other cases, it is desired to detect and identity the presence of related compounds (e.g. metabolites of the drug), and the instrument is used in a mode in which the entire product spectrum is obtained, or in which a spectrum of those precursor ions which form a specific (characteristic) product or which lose a characteristic neutral molecule (i.e. there is a fixed mass difference provided between the precursor ion and the selected product ion) is produced. The former scan mode is called a Precursor Ion Scan, and the latter is called a Neutral Loss Scan.
A common type of tandem mass spectrometer is a triple quadrupole. This is composed of a quadrupole mass filter (commonly designated as Q1) followed by a low pressure collision cell (again, commonly designated as Q2, as it usually includes a similar quadrupole rod set) filled with nitrogen or argon at a pressure of a few millitorr, followed by a second mass filter (Q3), followed by an ion detector. Ions must pass through the first mass filter, collision cell and second mass filter in order to be detected. In a Product Scan Mode, Q1 is tuned to the precursor m/z value of interest, and the second mass filter (Q3) is scanned to record an MS/MS spectrum. In a Precursor Scan Mode, Q1 is scanned while Q3 is fixed at a product ion of interest. In a Neutral Loss Scan mode, both quadrupoles are scanned with a fixed mass difference between them.
A second type of tandem mass spectrometer is a quadrupole/time-of-flight system (QqTOF). In this instrument, Q1 and Q2 are followed by a time-of-flight mass spectrometer, which provides higher mass resolution and mass accuracy than a quadrupole mass spectrometer. (In the acronym QqTOF, Q designates Q1 and q designates Q2, the lower case indicating that it is not a mass analyzer and TOF indicates a time-of-flight section.) It also allows quasi-simultaneous detection of all ions in an ion pulse which is admitted to the TOF section.
Another known and different type of tandem mass spectrometer is a quadrupole ion trap. In this device, all mass analysis is performed on ions which are trapped within a fixed volume (within quadrupole electrodes inside a vacuum system). Ions are trapped within a radio-frequency quadrupole field, and by changing the amplitude and waveform applied to the surrounding electrodes, ions can be isolated (to remove all but a selected m/z), fragmented (by collisions with a low density gas which fill the device), and then scanned to record a mass spectrum. Because all of the events occur in the same region of space, but sequentially in time (first filling the trap with ions, then isolating the precursor ion, then fragmenting the precursor ions, then recording the mass spectrum of the products), the ion trap is sometimes referred to as “tandem in time” as opposed to a triple quadrupole which is “tandem in space”.
Another related type of tandem mass spectrometer is a Fourier Transform Mass Spectrometer (FTMS). This is composed of a Penning Ion Trap, with the trapping region formed by the combined action of a strong magnetic field and a static electrostatic field. As in a quadrupole ion trap, MS/MS can be performed by the “tandem-in-time” process.
MS/MS/MS (or MS3) is an extension of the technique of MS/MS. In this case, fragment ions of a fragment ion are formed (second generation products). For example, the m/z 195 product ion from Reserpine can be selected and fragmented. This can provide further detailed information of the structure of m/z 195, or can be used as a second level confirmation of the identity of Reserpine (by requiring that the Product Ion Spectrum of 609, and Product Ion Spectrum of the 195 fragment, both match that of Reserpine). From an instrumental point of view, MS/MS/MS requires that the precursor ion be isolated (eliminating all other m/z values), then fragmented, then the m/z 195 ion isolated (eliminating all other fragment ions), then the 195 ion fragmented and its spectrum recorded. The process can, in principle, be repeated to perform any desired level of MSn; however since signal-to-noise (S/N) decreases at each stage, it is usually only common to perform MS3.
MS3 is usually only possible in ion trap or FTMS mass spectrometers (see Strife et al in Rapid Commun. Mass Spectrom. 14, 250–260, 2000.). In an ion trap, for example, ions from the source are trapped, and all but the precursor ion of interest is expelled or ejected from the trap. As mentioned above, this is done by using an auxiliary voltage with a wide range of frequencies to resonantly excite the motion of all ions except the one to be kept in the trap, until all other m/z ions are ejected. The precursor ion is then fragmented by gently exciting the motion of the precursor ion, until it fragments through multiple collisions with the low density background gas. All of the products are trapped. Then, the isolation step is repeated, ejecting all except the product ion of interest (for example, m/z 195 product of Reserpine). The motion of the product ion is then excited until it fragments, again trapping all of the products. The population of product ions is then scanned out of the trap and detected in order to product a mass spectrum. The entire cycle described constitutes MS/MS/MS of 609/195/products. A similar process is used in FTMS in order to perform MS/1MS/MS. In both instruments, the process can be repeated to fragment one of the trapped second-generation product ions, in order to do MS4 and higher order experiments.
In other types of tandem mass spectrometers, such as triple quadrupoles and QqTOF instruments, which perform MS/MS by means of two mass spectrometers which are separated in space, higher orders of MS can only normally be done by adding another collision cell and another mass spectrometer. For example, Beaugrand et. al. (Proc. 34th ASMS Conference on Mass Spectrometry and Allied Topics, 1986, p220) describe a pentaquadrupole system for performing MS/MS/MS and related experiments. However, such configurations are complex and expensive, and are not commonly available. They also cannot reasonably be extended to higher levels of MSn, due to the complexity and cost of the instrument and poor signal-to-noise ratios.
There are some recent methods which have been developed in order to allow MSn to be performed in a triple quadrupole or QqTOF-type of tandem mass spectrometer. For example, a co-pending Canadian patent application 2,274,186 by Lisa Cousins and Bruce Thomson, filed Jun. 10, 2000 and assigned to the assignee of the present application, describes a method of producing MS/MS/MS spectra by employing one or more excitation processes to the ion beam as it passes through the collision cell, and turning the excitation source on and off rapidly in order to statistically correlate second and third generation product ions with their precursors. This technique is relatively simple to implement, but it does not provide true MS/MS/MS because the precursor ions at each stage are not isolated from others. Therefore at low sample concentrations, the S/N of this method can be poor. It also does not allow unit mass resolution of the precursor ions, since the excitation signal can excite neighboring ions (within a few m/z values) to fragment, which complicates the spectrum. In addition, the method of excitation requires that a AC voltage supply be provided for the collision cell in order to radially excite the ions. This requires extra cost and complexity.
A further limitation of this method is that ion fragmentation for the second fragmentation stage is performed by radially exciting the motion of the trapped ions until they fragment through collisions. This excitation has to be carefully controlled in order that the ions not be excited too far and hit the rods. Generally, this type of excitation causes ions to be gently heated or excited, and to fragment through the lowest energy channels. The fragmentation spectrum which results is often different from the standard CAD spectrum obtained in a triple quadrupole or QqTOF mass spectrometer, and some high energy fragments may not be observed.
In U.S. Pat. No. 6,011,259, Whitehouse et al have described a method for MS/MS/MS in an orthogonal TOF system, by trapping ions in an RF quadrupole (containing a buffer gas at low pressure) in front of the TOF, and using auxiliary excitation to perform the steps of isolation and fragmentation in the 2-D trap. This is very analogous to the techniques used in a 3-D Paul trap as described above. After one or more steps of isolation and fragmentation (for MS/MS or MSn), the ions are released from the trap for mass analysis in the TOF mass spectrometer. In PCT Application PCT/CA99/01142 Douglas et. al. describe a similar technique in the collision cell of a QqTOF system.
Another recently described method is in co-pending U.S. provisional application 60/219,684 by James Hager and Jeff Plomley, in which MS/MS/MS is provided in a configuration, and in which ions are trapped in a collision cell (2-D quadrupole), and then the precursor ion mass is isolated by changing the RF voltage on the collision cell. The isolated precursor ion is then ejected into the next quadrupole (Q3), and is fragmented during the passage into Q3 by a few collisions with the gas emanating from the collision cell. The product ions are trapped in Q3, and then mass selectively scanned out of Q3. The entire process provides MS/MS/MS capabilities. However, the resolution provided by the method of isolation of the primary product ions (by changing the RF level on the collision cell) is rather low (for example a window of a few m/z values in width). Also, the efficiency of fragmentation by passage through the region between the quadrupoles is only about 40%, and it is limited to MS3, without the possibility of higher orders of MSn.
The methods described above (except the last one) all require auxiliary AC voltages to be applied to an RF-only quadruple, in order to isolate and/or fragment the ions. This requires extra cost and complexity, and requires careful control of this voltage and frequency in order to accurately isolate the correct m/z value. Using this method of isolation it is also difficult to achieve unit mass resolution.